Cell-controlled perfusion in continuous culture

ABSTRACT

Methods of protein production in continuous perfusion mammalian cell culture bioreactors are provided. Methods for continuous perfusion culture by allowing cells to self-regulate the rate of addition of perfusion medium to the bioreactor via a pH change are presented. Compositions comprising the perfusion medium as well as the process advantages of using hi-end pH control of perfusion or HIPCOP are also presented.

RELATED APPLICATIONS

This application is a divisional from pending U.S. Ser. No. 15/577,415,filed Nov. 28, 2017, which in turn is a national stage application under35 U.S.C. § 371 from PCT/US2016/034570, filed May 27, 2016, now expired,which in turn claims the priority benefit of now-expired U.S. SerialNos. 62/168,297, filed May 29, 2015; 62/199,388, filed Jul. 31, 2015 and62/246,774, filed Oct. 27, 2015, each of which are incorporated hereinby reference in their entireties.

FIELD OF THE SUBJECT TECHNOLOGY

The subject technology relates to methods of protein production incultured animal cells, preferably mammalian cells, using continuousperfusion cell culturing in a bioreactor.

BACKGROUND OF THE SUBJECT TECHNOLOGY

Microorganisms or single cells of multicellular species are grown invarious ways, including batch, fed-batch, continuous cultures,continuous cultures with cell retention (perfusion) or combinationsthereof. Batch cultures require that a microbe or cell, for example,yeast, bacteria or fungal inoculums, is grown in a closed culture systemon a limited or constant amount of growth medium. In batch cultures,there are a number of growth phases that the microbe passes throughbefore cell death due to consumption of nutrients in the medium andproduction of toxic metabolites. Fed-batch cultures are similar to batchculture methods except that the batch culture is fed with nutrientmedium in a fermentor or bioreactor without removing growth culture orgrowth products. As expected, the volume of the culture fluid in thebioreactor increases over time but typically high cell densities areachieved in this method of culture as compared to batch culture.Continuous cultures, in which the bioreactor volume remains constant andfresh medium is added and culture fluid is removed continuously, areanother operational mode. In such a culture additional cell division islikely to occur, particularly if there is a continuous removal of cellsfrom the bioreactor. Continuous cultures can also employ a cellretention device which retains all or nearly all cells within thebioreactor. When cell retention is employed cell densities can be muchhigher in the bioreactor as higher rates of medium flow in and out ofthe bioreactor (higher perfusion rates) are possible without the risk ofcell washout. It is also possible to combine modes of bioreactoroperation.

Due to the limits of feed volume addition and the problem of amino acidcounter ion, miscellaneous osmolyte, and cell growth inhibitoraccumulation, the fed-batch mode of bioreactor operation of cellproduction, for example, Chinese hamster ovaries (CHO) cell production,of protein therapeutics is inherently limited with respect to the celldensities and productivities that are achievable. Lactic acid, a growthinhibitor secreted during cell growth, is especially problematic. Lacticacid is produced by the breakdown of glucose in the cells and causes thebulk pH to drop as its concentration increases. Although lactic acid canbe neutralized to lactate with the addition of a base titrant duringcell culture, the addition of base results in large changes in osmoticstrength of the growth medium due to ion accumulation. Both the rise inosmotic strength due to lactate accumulation, and the lactate ion itselfcan ultimately slow cell growth and can cause loss of cell productivity.Other efforts to reduce lactic acid formation include limiting glucoseconcentration, substituting alternative six-carbon sugars such asgalactose, fructose, or mannose for glucose, and/or manipulating certainenzymes or substrate membrane transporters during cell culturing. Morerecently, high-end pH-controlled delivery of glucose has been shown tolimit lactic acid secretion during fed-batch culture (Luan et al., U.S.Pat. No. 7,429,491 B2 and Gagnon et al., Biotechnol. Bioeng., 2011; 108:1328-1337).

Continuous or perfusion culture with cell retention can overcome some ofthese limitations, but suffers from the disadvantages of large volumemedia consumption, long times to reach peak cell densities andcomplications with cell retention devices. Therefore, there stillremains a need for an alternative culture method that overcomes thelimitations associated with fed-batch and/or conventional continuousperfusion cultures.

SUMMARY OF THE SUBJECT TECHNOLOGY

The goal of this subject technology is to overcome current limitationsin continuous perfusion cell culture and provide alternative methods tooptimize cell growth and viability.

A method of continuous culture is described that overcomes many of thelimitations outlined above and the method utilizes a unique technologywhich allows cells to control their own rate of perfusion withcontinuous feedback, i.e., self-regulating cells. The terms “hi-end pHcontrol of perfusion or HIPCOP” are used to describe the process bywhich cells control their own rate of perfusion. Volumetricproductivities of equal to about, or greater than, 1 gram/L/day formoderate specific productivity cell lines are achieved with very modestmedium volumes, comparatively simple bioreactor operations, and a batchlength that fits in a standard fed-batch window. The volumetricproductivity achieved is more than double what one might achieve withthe same cell line in an optimized fed-batch culture.

In its broadest aspect, the subject technology relates to a continuousperfusion culture process comprising monitoring pH in a cell culture inperfusion bioreactor with a pH controller or sensor, activating a mediumperfusion pump delivering fresh medium and a permeate perfusion pumpremoving a nearly equivalent volume of permeate when the pH increasesabove a setpoint or predetermined value, and deactivating the mediumperfusion pump and permeate perfusion pump when the pH decreases below asetpoint or predetermined value. In other words, in an embodiment, thesubject technology relates to a continuous perfusion culture process,including: (a) monitoring pH in a cell culture with a pH sensor; (b)delivering fresh medium and removing permeate when the pH is above apredetermined value; and (c) deactivating the medium delivery and thepermeate removal when the pH decreases below the predetermined value. Inessence, the pH change in the cell culture medium is a result of thecell metabolism which then triggers the perfusion process.Alternatively, the method may be described as a cell-controlledperfusion process without a need to measure glucose concentration. ThepH trigger for turning on and off the perfusion pumps, fresh medium andpermeate pumps, is set at a predetermined value. This predeterminedvalue is about pH 7 (e.g., between 6.8-7.4). The medium comprisesglucose, L-lactate and a specified ratio of amino acid to glucose.According to this subject technology, the activation and deactivation ofthe medium perfusion pump and the permeate perfusion pump may occursimultaneously or independently. Also, the permeate may be cell-free ormay contain cells.

The described method also comprises adding L-lactate to a freshperfusion medium used in the continuous culture process. In anembodiment, the L-lactate present in the perfusion medium is in anamount of about 0.1 g/L to about 7.0 g/L. More preferably, the L-lactatepresent in the perfusion medium is in an amount of about 1 to about 4g/L, about 1 to about 3 g/L, or about 1 to about 2.5 g/L. In a preferredembodiment, the L-lactate is sodium L-lactate or potassium L-lactate.

In another embodiment, instead of having L-lactate in the perfusionmedium, additional sodium bicarbonate is delivered to the fresh mediumor to perfusion bioreactor when the pH of the perfusion culture goesabove the predetermined value (i.e., pH 7 or a pH of 6.8-7.4). A typicalcell culture medium contains about 1.0 to 2.5 g/L of sodium bicarbonate.In this embodiment, however, an additional 1 to 3 grams of sodiumbicarbonate is added to the perfusion medium for every 1 liter ofperfusion media added to the perfusion cell culture bioreactor when thepH of the cell culture is above pH 6.8 or above pH 7 or above pH 7.4 orabove any pH within the range of 6.8 to 7.4. Once the pH drops below thepredetermined value, the delivery of sodium bicarbonate is stopped ordiscontinued. Any other physiologically acceptable base (e.g., sodiumcarbonate, potassium carbonate, HEPES buffer, or the like), which isknown to one of ordinary skill in the art, may be used in place ofsodium bicarbonate so long as such base is added to the perfusionbioreactor slowly and at a rate that would provide an upward influenceon the pH in a manner similar to that which would occur by theconsumption of lactate from the perfusion medium as lactic acid. Forexample, instead of sodium bicarbonate being added to the perfusionmedium, sodium carbonate can be added to the perfusion bioreactor slowlywhen the pH is above the predetermined value. In an exemplaryembodiment, the sodium carbonate is added to the perfusion bioreactorsuch that 1 molar carbonate enters the perfusion bioreactor at a rate of8.7 mL per 1 liter of perfusion medium utilized.

The fresh medium in this method requires at least glucose and aminoacids. In an embodiment, the concentration of glucose is in the range ofabout 0.5 to about 40 g/L. In another embodiment, the fresh mediumcontains L-lactate. The concentration of L-lactate is in the range ofabout 0.1 and about 7.0 g/L. The ratio of moles of glucose to aminoacids is between about 0.25 and 1.0. In another embodiment, the freshmedium further contains sodium bicarbonate, in addition to or in placeof L-lactate, in an amount of about 2 to 5.5 g/L. In another embodiment,the fresh perfusion medium contains both L-lactate (in an amount ofabout 0.1 to 7 g/L) and sodium bicarbonate (in an amount of about 2 toabout 5.5 g/L) such that the cells will be able to control theirperfusion rate over the entire continuous perfusion period according tothe subject technology.

Thus, in an aspect, the subject technology relates to a perfusionculture process, including: (a) monitoring pH in a cell culture in aperfusion bioreactor with a pH sensor; (b) delivering fresh medium andremoving permeate when the pH is above a predetermined value; and (c)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value. In one or more embodiments related,directly or indirectly, to this aspect to each other, the fresh mediumincludes L-lactate; the L-lactate is present in the fresh medium in anamount of about 0.1 g/L to 7.0 g/L; the L-lactate is present in thefresh medium in an amount of about 1 to 4 g/L; the L-lactate is presentin the fresh medium in an amount of about 1 to 3 g/L; the L-lactate ispresent in the fresh medium in an amount of about 1 to 2.5 g/L;alternatively or in addition to L-lactate, additional sodium bicarbonateis added to the perfusion medium or the perfusion bioreactor in anamount of about 1 to about 2.5 g/L such that the total sodiumbicarbonate in the culture medium is about 2 to about 5.5 g/L andwherein the additional sodium bicarbonate is delivered to the perfusionbioreactor when the pH is above the predetermined value; the additionalsodium bicarbonate is added to the perfusion bioreactor such that 1molar carbonate enters the perfusion bioreactor at a rate of 8.7 mL per1 liter of perfusion medium utilized; alternatively or in addition toL-lactate, any other physiologically acceptable base such as sodiumcarbonate, potassium carbonate, or the like is added to the perfusionbioreactor in an amount that would provide an upward influence on the pHin a manner similar to that which would occur by the addition ofL-lactate; the fresh medium includes: (a) glucose; (b) L-lactate and/oradditional sodium bicarbonate; and (c) amino acids; the fresh mediumincludes: (a) between about 0.5 to about 40 g/L glucose; (b) betweenabout 0.1 to about 7 g/L L-lactate and/or between about 2 to about 5.5g/L sodium bicarbonate; and (c) amino acids in amole-of-glucose-to-mole-of-amino-acids ratio of between about 0.25 toabout 1.0; the fresh medium includes glucose in an amount equal to about70 mM of amino acids and about 5.3 grams of glucose per liter of medium;the fresh medium includes glucose at an amino-acids-(inmM)-to-glucose-(in g/L) ratio selected from the group consisting ofabout 60 to about 4.2; about 90 to about 8; about 100 to about 12; about120 to about 13; about 240 to about 42 and about 380 to about 70 perliter of medium; the predetermined pH value is about pH 7 or is about6.8 to about 7.4; a measurement of glucose concentration in the cellculture or addition of glucose to the cell culture by a glucose pump isnot required.

In another aspect, the subject technology relates to a method forachieving rapid cell growth in a perfusion culture process, comprising:(a) monitoring pH in a cell culture in a perfusion bioreactor with a pHsensor; (b) delivering fresh medium and removing permeate when the pH isabove a predetermined value; and (c) deactivating the medium deliveryand the permeate removal when the pH is below the predetermined value.In one or more embodiments related, directly or indirectly, to thisaspect to each other, the fresh medium comprises L-lactate; L-lactate isin an amount of about 0.1 g/L to 7.0 g/L; alternatively or in additionto L-lactate, additional sodium bicarbonate is added to the perfusionbioreactor in an amount of about 1 to about 2.5 g/L such that the totalsodium bicarbonate in the culture medium is about 2 to about 5.5 g/L andwherein the additional sodium bicarbonate is delivered to the perfusionbioreactor when the pH is above the predetermined value; the additionalsodium bicarbonate is added to the perfusion bioreactor such that 1molar carbonate enters the perfusion bioreactor at a rate of 8.7 mL per1 liter of perfusion medium utilized; alternatively or in addition toL-lactate, any other physiologically acceptable base such as sodiumcarbonate, potassium carbonate, or the like is added to the perfusionbioreactor in an amount that would provide an upward influence on the pHin a manner similar to that which would occur by the addition ofL-lactate; the fresh medium includes: (a) glucose; (b) L-lactate and/oradditional sodium bicarbonate; and (c) amino acids; the fresh mediumincludes: (a) between about 0.5 to about 40 g/L glucose; (b) betweenabout 0.1 to about 7 g/L L-lactate and/or between about 2 to about 5.5g/L sodium bicarbonate; and (c) amino acids in amole-of-glucose-to-mole-of-amino-acids ratio of between about 0.25 toabout 1.0; the fresh medium includes glucose in an amount equal to about70 mM of amino acids and about 5.3 grams of glucose per liter of medium;the fresh medium includes glucose at an amino-acids-(inmM)-to-glucose-(in g/L) ratio selected from the group consisting ofabout 60 to about 4.2; about 90 to about 8; about 100 to about 12; about120 to about 13; about 240 to about 42 and about 380 to about 70 perliter of medium; the predetermined pH value is about pH 7 or is about6.8 to about 7.4; a measurement of glucose concentration in the cellculture or addition of glucose to the cell culture by a glucose pump isnot required; a viable cell density of about 60×10⁶/mL is achievedwithin 4 days.

Another aspect of the subject technology relates to a hybridperfusion/fed-batch culture process. This process comprising using thecontinuous perfusion process described above in combination with afed-batch process. According to the subject technology, the processwould run in perfusion mode for 4 to 6 days prior to being switched tofed-batch mode for an additional 4 to 8 days. By using this hybrid mode,volumetric productivity of equal to about or greater than 1.0 gram/L/dayis achieved. In this embodiment, the continuous perfusion cultureprocess includes steps (a) monitoring pH in a cell culture with a pHsensor; (b) delivering fresh medium and removing permeate when the pH isabove a predetermined value; (c) and deactivating the medium deliveryand the permeate removal when the pH is below the predetermined value.

Another aspect of the subject technology relates to a continuousperfusion bioreactor which consists of two significantly differentphases. The first phase includes the initial continuous phase asmentioned above (ramp up of cell density and perfusion rate usingHIPCOP) utilizing a comparably dilute medium, followed by a second phaseof perfusion in which the perfusion rates are significantly reduced byutilization of a highly concentrated perfusion medium. By using thistwo-phase perfusion system, volumetric productivities of equal to aboutor greater than 1.5 grams/L/day is achieved while using very modestvolumes of perfusion medium. No significant cell bleed (removal of cellsfrom the bioreactor) occurs in the examples presented in thisapplication.

Another aspect of the subject technology relates to the use of a diluentliquid that is added to the bioreactor during the later stages of theperfusion culture when the concentrated perfusion medium is being addedto the bioreactor. In one embodiment, such a diluent liquid is asolution of saline of appropriate concentration. Because thelong-distance transport of liquid nutrient medium can incur manydifficulties (cost of transport, maintenance of sterility, temperaturecontrol) there is significant value in using highly concentratedperfusion medium in an industrial setting. Such medium allows forperfusion rates as low as 0.05-0.30 reactor volumes per day for theconcentrated perfusion medium. In a related embodiment, in such abioreactor system it is necessary to flush product material out of thebioreactor, particularly if a continuous downstream process is linkeddirectly to capture the continuously delivered upstream harvest material(e.g., the material coming through the hollow fiber cell retentionsystem), and if the protein being produced is highly labile.

In another aspect, the subject technology relates to a hybrid cultureprocess, including a first continuous perfusion culture process followedby a second continuous perfusion culture process, wherein the firstcontinuous perfusion culture process includes steps (a) monitoring pH ina cell culture with a pH sensor; (b) delivering fresh medium andremoving permeate when the pH is above a predetermined value; (c) anddeactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value.; and wherein the second continuousperfusion culture process includes steps of (a) adding a concentratedmedium; and (b) adding a diluent. In a related embodiment, the hybridculture process optionally includes an intervening fed-batch step. Inthis embodiment, the first continuous perfusion culture process iscarried out first followed by a fed-batch step which is then followed bythe second continuous perfusion culture process. In one or moreembodiments related, directly or indirectly, to this aspect to eachother, the continuous perfusion culture process is the first cultureprocess; the continuous perfusion culture process is followed by afed-batch process; the continuous perfusion culture process is followedby a second continuous perfusion culture process; the second continuousperfusion culture process comprises the steps of: (a) adding aconcentrated medium; and (b) adding a diluent; the concentrated mediumcomprises 600 millimolar amino acids, 90 grams/liter glucose, 0 g/Lsodium L-lactate; the diluent is selected from the group consisting ofsaline and water; the saline or water normalizes osmotic strength ofculture medium to between about 0 to 250 and/or 250 to 350 mOsm/kg; thesaline comprises 2.0 g/L sodium bicarbonate, 2.4 g/L polyvinyl alcohol,20 mM potassium chloride, and 80 mM sodium chloride; the hybrid cultureprocess results in volumetric productivity of greater than 1 gramcells/L/day; the hybrid culture process is performed in a culture volumeof about 50 and about 150 L; the culture volume is about 70 L.

In another aspect, the subject technology relates to a bioreactor forconducting a continuous perfusion culture process, wherein thecontinuous perfusion culture process includes steps of: (a) monitoringpH in a cell culture in a perfusion bioreactor with a pH sensor; (b)delivering fresh medium and removing permeate when the pH is above apredetermined value; (c) and deactivating the medium delivery and thepermeate removal when the pH is below the predetermined value. In one ormore embodiments related, directly or indirectly, to this aspect to eachother, the fresh medium comprises: (a) glucose; (b) L-lactate; and (c)amino acids; L-lactate is in an amount of about 0.1 g/L to 7.0 g/L;alternatively or in addition to L-lactate, additional sodium bicarbonateis added to the perfusion bioreactor in an amount of about 1 to about2.5 g/L such that the total sodium bicarbonate in the culture medium isabout 2 to about 5.5 g/L and wherein the additional sodium bicarbonateis delivered to the perfusion bioreactor when the pH is above thepredetermined value; the additional sodium bicarbonate is added to theperfusion bioreactor such that 1 molar carbonate enters the perfusionbioreactor at a rate of 8.7 mL per 1 liter of perfusion medium utilized;alternatively or in addition to L-lactate, any other physiologicallyacceptable base such as sodium carbonate, potassium carbonate, or thelike is added to the perfusion bioreactor in an amount that wouldprovide an upward influence on the pH in a manner similar to that whichwould occur by the addition of L-lactate; the fresh medium includes: (a)glucose; (b) L-lactate and/or additional sodium bicarbonate; and (c)amino acids; the fresh medium includes: (a) between about 0.5 to about40 g/L glucose; (b) between about 0.1 to about 7 g/L L-lactate and/orbetween about 2 to about 5.5 g/L sodium bicarbonate; and (c) amino acidsin a mole-of-glucose-to-mole-of-amino-acids ratio of between about 0.25to about 1.0; the fresh medium includes glucose in an amount equal toabout 70 mM of amino acids and about 5.3 grams of glucose per liter ofmedium; the fresh medium includes glucose at an amino-acids-(inmM)-to-glucose-(in g/L) ratio selected from the group consisting ofabout 60 to about 4.2; about 90 to about 8; about 100 to about 12; about120 to about 13; about 240 to about 42 and about 380 to about 70 perliter of medium; the predetermined pH value is about pH 7 or is about6.8 to about 7.4; a measurement of glucose concentration in the cellculture or addition of glucose to the cell culture by a glucose pump isnot required.

In another aspect, the subject technology relates to a protein ofinterest produced by a method including: (a) culturing cells comprisinga gene that encodes the protein of interest in a perfusion cell culturebioreactor under conditions that allow production of the protein ofinterest including: (i) monitoring pH in the cell culture in a perfusionbioreactor with a pH sensor; (ii) delivering fresh medium and removingpermeate when the pH is above a predetermined value; and (iii)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value, (b) harvesting the protein of interestfrom the cell culture bioreactor. In one or more embodiments related,directly or indirectly, to this aspect to each other, the fresh mediumincludes: (a) glucose, (b) L-lactate, and (c) amino acids; L-lactate isin an amount of about 0.1 g/L to 7.0 g/L; alternatively or in additionto L-lactate, additional sodium bicarbonate is added to the perfusionbioreactor in an amount of about 1 to about 2.5 g/L such that the totalsodium bicarbonate in the culture medium is about 2 to about 5.5 g/L andwherein the additional sodium bicarbonate is delivered to the perfusionbioreactor when the pH is above the predetermined value; the additionalsodium bicarbonate is added to the perfusion bioreactor such that 1molar carbonate enters the perfusion bioreactor at a rate of 8.7 mL per1 liter of perfusion medium utilized; alternatively or in addition toL-lactate, any other physiologically acceptable base such as sodiumcarbonate, potassium carbonate, or the like is added to the perfusionbioreactor in an amount that would provide an upward influence on the pHin a manner similar to that which would occur by the addition ofL-lactate; the fresh medium includes: (a) glucose; (b) L-lactate and/oradditional sodium bicarbonate; and (c) amino acids; the fresh mediumincludes: (a) between about 0.5 to about 40 g/L glucose; (b) betweenabout 0.1 to about 7 g/L L-lactate and/or between about 2 to about 5.5g/L sodium bicarbonate; and (c) amino acids in amole-of-glucose-to-mole-of-amino-acids ratio of between about 0.25 toabout 1.0; the fresh medium includes glucose in an amount equal to about70 mM of amino acids and about 5.3 grams of glucose per liter of medium;the fresh medium includes glucose at an amino-acids-(inmM)-to-glucose-(in g/L) ratio selected from the group consisting ofabout 60 to about 4.2; about 90 to about 8; about 100 to about 12; about120 to about 13; about 240 to about 42 and about 380 to about 70 perliter of medium; the predetermined pH value is about pH 7 or is about6.8 to about 7.4; a measurement of glucose concentration in the cellculture or addition of glucose to the cell culture by a glucose pump isnot required.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the claimedmethods, apparatuses, and systems are better understood when thefollowing detailed description is read with reference to theaccompanying drawings:

FIG. 1 shows a diagram of a perfusion apparatus.

FIG. 2A illustrates a pictorial representation of hypothesized sequenceof events occurring in the bulk cell culture fluid during the growthphase of a bioreactor utilizing the HIPDOG control scheme for limitinglactic acid accumulation in fed-batch culture.

FIG. 2B illustrates a pictorial representation of hypothesized sequenceof events occurring in the bulk cell culture fluid during the growthphase of a bioreactor utilizing the HIPCOP control scheme forcell-controlled perfusion rate ramp up in continuous perfusion culture.

FIG. 3 (A-E) illustrates the results of CHO cell line A growth in a 2 Lbioreactor (as described in Example 1). Open symbols represent resultswhen using the HIPCOP technology. Solid symbols represent thenon-glucose limited condition. Perfusion is ‘self-initiated’ when the pHof the culture begins to rise as lactic acid is first removed from thebulk culture causing a rise in pH. FIG. 3A shows viable cell density (E5cells/mL) over time (days); FIG. 3B shows viability (%) over time(days); FIG. 3C shows glucose (g/L) over time (days); FIG. 3D showsL-lactate concentration (g/L) over time (days); and FIG. 3E showsosmolality (mOsm/kg) over time (days).

FIG. 4 (A-C) illustrates the results of CHO cell line B growth in a 2 Lbioreactor using HIPCOP method of controlling perfusion rate (asdescribed in Example 1). FIG. 4A shows either viable cell density (E5cells/mL) or viability (%) over time (days); FIG. 4B shows metaboliteconcentration (g/L) over time (days); FIG. 4C shows osmolality (mOsm/kg)over time (days).

FIG. 5 (A-C) illustrates the results of the CHO cell line A growth in a150 L bioreactor (70 L working volume) using the HIPCOP method forcontrolling perfusion rate (as described in Example 2). FIG. 5A showseither viable cell density (E5 or 10⁵ cells/mL) or viability (%) overtime (days) of CHO cell line A in a 70-liter working volume pilot scalebioreactor using the HIPCOP method of controlling perfusion rate; FIG.5B shows metabolite concentration (g/L) over time (days); FIG. 4C showsosmolality (mOsm/kg) over time (days).

FIG. 6 (A-E) illustrates the results of four differentglutamine-sythetase expression system CHO cell lines (cell lines A, B,C, and D) producing IgG antibodies grown in 2 L bioreactors using theHIPCOP perfusion technology until day 4 and then converting to afed-batch operational mode for the remainder of the 12-day culture. FIG.6A shows viable cell density (E6 or 10⁶ cells/mL), FIG. 6B shows percentviable cells as measured by trypan blue exclusion, FIG. 6C showsL-lactate concentration over time, FIG. 6D shows osmotic strength overtime (mOsm/kg), and FIG. 6E shows product titer (IgG antibody)accumulation over time.

FIG. 7 illustrates the day-12 product titer (grams/liter of IgGantibody) of the hybrid perfusion/fed-batch process that used HIPCOPduring the cell expansion phase in comparison to the optimized titer ofthe 12-day fed-batch process.

FIG. 8 illustrates the modest volumes of perfusion medium (expressed inreactor volumes of perfusion medium) that are used during the HIPCOPphase of perfusion to achieve the high cell densities and productivitiesindicated in FIGS. 6A-E and FIG. 7 . This table also shows thecalculated maximum volumetric productivity which can be obtained fromFIG. 7 by dividing by the number of days of culture at any time point inthe culture.

FIG. 9A-F illustrates the cell densities, cell viabilities, L-lactateconcentrations, osmotic strength, product titer, and rates of perfusionas a function of time for a 2 L continuous-perfusion culture of IgGantibody-producing CHO cell line A that uses HIPCOP initially during thecell expansion phase (days 1-6) and a manually adjusted perfusion ratefrom day 6 to the end of the culture. In FIG. 9F the total perfusionrate is indicated by solid diamonds, the concentrated perfusion mediumby solid squares, and the saline diluent by open squares.

FIG. 10 is a table listing the overall volumetric productivity ascalculated assuming all product of interest is captured from a finalharvest of the bioreactor and the permeate collected from days 6-17using the entire length of the process (17 days) as the divisor. Alsolisted are the total volumes of initial perfusion medium (days 1-6),concentrated nutrient perfusion medium (days 6-17) and saline diluent(days 6-17) used in the experiment of FIG. 9A-F.

FIG. 11 is a bar graph illustrating the increases in overall processvolumetric productivity when compared with an optimized fed-batchprocess (first bar from left, a 12-day process) that can be achievedusing the HIPCOP technology paired with either a hybrid perfusionfed-batch operational mode (2^(nd) bar from left, a 12-day process), ora continuous perfusion operational mode (high-intensity, low volumeperfusion, a 17-day process, 3^(rd) bar from left). All data in thisfigure was collected using cell line A as described in earlier figuresand in the examples. The overall process volumetric productivitycalculations assume that product leaving the bioreactor system in thepermeate, and that remaining in the bioreactor can be utilized. Thematerial in the bioreactor presumably would be captured by a finalharvest step at the end of the process. The calculations do not includethe product lost to the permeate stream during the expansion phase whilecomparatively dilute perfusion medium is being utilized (while theHIPCOP technology is in operation, day 0-4 for the hybrid perfusionfed-batch process, and day 0-6 for the continuous perfusion process).Furthermore, the calculations utilize the entire batch length (frominoculation to termination at harvest) as the divisor.

FIG. 12 (A-C) illustrates the results of a DG-44 derived CHO cell line Eproducing a recombinant protein of ˜130 kDa. Growth occurs in 3 Lbioreactor using HIPCOP method of controlling perfusion rate (asdescribed in Example 1). FIG. 12A shows either viable cell density (E6cells/mL) or viability (%) over time (days); FIG. 12B shows metaboliteconcentration (g/L) over time (days); FIG. 12C shows osmolality(mOsm/kg) over time (days).

FIG. 13 (A-C) illustrates the results of a GS-CHO cell line B producinga recombinant immunoglobulin G. Growth occurs in 3 L bioreactor usingHIPCOP method of controlling perfusion rate (as described in Example 1).FIG. 13A shows either viable cell density (E6 cells/mL) or viability (%)over time (days); FIG. 13B shows metabolite concentration (g/L) overtime (days); FIG. 13C shows osmolality (mOsm/kg) over time (days).

FIG. 14 (A-E) illustrates the results of a GS-CHO cell line B producinga recombinant immunoglobulin G. Culture was maintained in a 3 Lbioreactor using HIPCOP method of controlling perfusion rate (asdescribed in Example 1). FIG. 14A shows either viable cell density (E6cells/mL) or viability (%) over time (days); FIG. 14B shows metaboliteconcentration (g/L) over time (days); FIG. 14C shows daily averageperfusion rate (VVD) over time (days); FIG. 14D shows cell-specificperfusion rate (pL/cell/day) over time (days); FIG. 14E shows osmolality(mOsm/kg) over time (days).

DETAILED DESCRIPTION OF THE SUBJECT TECHNOLOGY

The subject technology relates to methods for continuous perfusionculture by allowing cells to self-regulate the rate of addition ofperfusion medium to the bioreactor via a pH change and where theperfusion medium comprises glucose, L-lactate (and/or sodiumbicarbonate) and a specified ratio of amino acids to glucose. Advantagesof this technology include increased protein production while optimizingprocess conditions such as using less liquid media.

The subject technology is, in part, based on the surprising discoverythat L-lactate, a potentially growth inhibiting compound, canadvantageously be added to the perfusion medium for cells to be able tocontrol their perfusion rate over the entire continuous perfusionperiod.

Definitions

The term “about” generally refers to a slight error in a measurement,often stated as a range of values that contain the true value within acertain confidence level (usually ±1 σ for 68% C.I.). The term “about”may also be described as an integer and values of ±20% of the integer.

The term “about pH 7” refers to pH 7±1 pH units. In an embodiment, theabout pH 7 refers to pH 7±0.2 pH units. In another embodiment, the aboutpH 7 refers to pH of 6.8 to 7.4. In another embodiment, the about pH 7refers to pH 7.10±0.025 pH units. For example, the pH setpoint (orpredetermined value) and deadband during perfusion may be set to7.10±0.025. At this value, the perfusion pump is triggered at thehigh-end of this range, e.g., a pH of 7.125. When the pH rises above7.125, the perfusion pump will turn on, and when the pH drops below7.125, the pump will turn off. A separate pump may be activated to addan alkaline solution when the pH drops below the low-end of the range at7.075.

Lactic acid or 2-hydroxypropanoic acid (CH₃CHOHCOOH) is an organic acidproduced and consumed by certain cells during culture. Lactic acid ischiral and has two optical isomers. One is known as L-(+)-lactic acid(chiral, (S)-lactic acid) and the other, its mirror image, isD-(−)-lactic acid (chiral, (R)-lactic acid). A mixture of the two inequal amounts is called DL-lactic acid.

L-lactate refers to an ester or salt of lactic acid. Lactate is aby-product of culture and is produced during cellular respiration asglucose is broken down. Esters of lactic acid may include, but are notlimited to, methyl L-lactate, ethyl L-lactate, butyl L-lactate,ethylhexyl L-lactate, lauryl L-lactate, myristyl L-lactate, or cetylL-lactates. Salts of L-lactates may refer to alkali metal L-lactatessuch as potassium L-lactate, sodium L-lactate, lithium L-lactate, orammonium L-lactate, as well as alkali earth metal L-lactates such ascalcium L-lactate, magnesium L-lactate, strontium L-lactate, or bariumL-lactates. In addition, L-lactates of other divalent, trivalent andtetravalent metals may include zinc L-lactate, aluminum L-lactate, ironL-lactate, chromium L-lactate, or titanium L-lactate. In accordance withthis subject technology, any ester or salt of lactic acid may be used.

The term “volumetric productivity” refers to the amount of materialproduced per volume per time of run. For mammalian cell culture, thisvalue may be reported as grams/L/day.

The term “permeate” refers to the liquid (including the spent medium andthe expressed protein) that leaves the bioreactor through one or morefilters, membranes or other cell retention devices. Depending on thetype of the filter/membrane or other cell retention device it passesthrough, permeate may be cell-free or may contain a residual amount ofcells.

Continuous Perfusion

During continuous perfusion culture of mammalian cells, medium isperfused through a culture while the cell mass is contained within thebioreactor by means of a cell retention device (FIG. 1 ). In asuspension culture system, the cell retention device is commonly afilter of some type, but numerous other methods can be employed (sonicseparation, inclined plane settling, external centrifuges, internalfilters such as spinning or oscillating, external hydrocyclones, etc.)including some devices that might not be completely ‘cell free’. As thecell mass continues to grow and increase in number and mass, the rate ofperfusion increases to remove metabolic by-products and supply necessarynutrients. The perfusion rate is commonly increased step-wise and inmany cases, is determined based on a calculation in which a specificratio of perfusion medium volume per time to cell number is maintained(CSPR, or cell-specific perfusion rate, often in nanoliters/cell/day,usually in the 0.05-0.5 nL/cell/day range). See Ozturk, Cytotechnology,1996; 2: 3-16 and Konstantinov et al., Adv. Biochem. Eng/Biotechnol.,2006; 101: 75-98. In some instances, the perfusion rate is set tocontrol the concentration of glucose or L-lactate (Konstantinov et al.,Biotechnol. Prog, 1996; January-February; 12(1): 100-9 and Ozturk etal., Biotechnol. Bioeng., 1997; February 20; 53(4): 372-8), or is basedon oxygen uptake rate measurements. See Feng et al., J. Biotechnol.,2006; April 20; 122(4): 422-430.

When compared with a fed-batch culture producing a similar amount ofproduct protein, continuous perfusion cultures typically utilize muchlarger volumes of cell culture medium. The larger volumes of medium arenot primarily needed to supply nutrients because the nutrient feeds canbe highly concentrated and added in small volumes as in a fed-batchculture. The larger volumes of medium used in perfusion culture aretypically employed to wash away metabolic byproducts of the cells. Cellsin a perfusion culture typically undergo higher levels of shear andother environmental insults that require at least some growth of cellsto make up for those that die. In addition, continuous cultures areexpected to operate for significantly longer lengths of time, severalweeks or even months, compared with a fed-batch culture that might lastat most 10-18 days. Consequently, cells in a continuous culture shouldbe maintained in a state that allows for at least some cell division.That state requires that the inhibitory metabolic byproducts are keptbelow a certain level.

The primary inhibitory compound generated by mammalian cells is lacticacid. This is particularly true when cells grow quickly. The lactic acidsuppresses pH and requires that base titrant be added so that the pH canremain in a range appropriate for cell growth. The lactic acid isneutralized to L-lactate and the ion sodium (or potassium) enters theculture in large amounts as the typical counter ion for the high pHtitrant. The L-lactate ion is itself inhibitory to growth atsufficiently high levels, but also the mere presence of the L-lactateand additional sodium ions significantly elevate the osmotic strength ofthe medium until it is outside of the normal physiological range of280-320 mOsm (see Gagnon et al., Biotechnol. Bioeng., 2011; 108:1328-1337). At a sufficiently high osmotic strength cell growth willslow and eventually productivity of a culture will also decrease.

Conventional continuous perfusion cultures often ramp up the perfusionrate in order to flush out accumulated L-lactate and keep cells growingat the start of a culture. This perfusion ramp up consumes large volumesof medium that might not otherwise be required if no L-lactate wereproduced.

When mammalian cells in culture are exposed to freely available glucose(concentrations perhaps above 2 mM) they typically produce high levelsof lactic acid and continue to maintain high glucose consumption rates.However, when the glucose levels are low (below 2 mM), mammalian cellswill cease the production of lactic acid and instead, will transportlactic acid from the bulk medium back across their membranes forconsumption. The net uptake of lactic acid from the bulk fluid causesthe pH of the culture to rise quickly. If the rise in pH triggers theslow addition of glucose to the culture, e.g., from a pump delivering anutrient solution containing glucose, then the pH on the high end can becontrolled to a near constant value at the same time as glucose (andpotentially other nutrients) is delivered to the culture, resulting inthe accumulation of lactic acid and its detrimental effects to theculture being suppressed. This process is the basis of previouslydescribed high-end pH delivery of glucose (HIPDOG) in FIG. 2A (Gagnon etal., Biotechnol. Bioeng., 2011; 108: 1328-1337, disclosing an experimentwith a fed-batch culture). High-end pH-controlled delivery of glucoseeffectively suppresses L-lactate accumulation, for example, in CHOfed-batch cultures. The HIPDOG process may be described as cellsself-determining their rate of glucose consumption because the cellsindicate a need for additional glucose by taking up lactic acid andtriggering a rise in the pH.

The HIPDOG fed-batch process may be extended to continuous perfusionculture of mammalian or CHO cells in the following manner. When glucoseconcentrations get too low in a continuous culture, the cells take uplactic acid, triggering a rise in pH. In a fed-batch culture a pumpdelivering a highly concentrated glucose solution is activated, i.e.,the glucose concentration of the feed commonly being between about 50and 500 g/L, whereas in continuous perfusion culture a pump may beactivated to deliver a perfusion medium containing a lower concentrationof glucose, for example between about 4 and 40 g/L glucose. Incontinuous perfusion culture the volume of the bioreactor is maintainedconstant. Therefore, any addition of perfusion medium coincides with anequivalent volume removal of culture medium from the bioreactor. Thiscan be accomplished, for example, by using a laboratory balance whichsignals a pump to remove permeate whenever the weight of the bioreactorexceeds the tare weight. When the glucose level in the culture becomesnon-limiting, the cells again excrete lactic acid which suppresses thebulk culture pH and deactivates the feed and permeate pumps that deliverand remove medium, respectively, to the culture. This cycle repeatsagain and again as the cells grow and metabolize. As the cell density ofthe culture increases, the cells are able to trigger the perfusion pumpto turn on more and more frequently, thereby ramping up their rate ofperfusion without any manual intervention.

During the above-described process, it was observed that the levels ofL-lactate in the bulk culture fluid dropped over time. As a result, itwas necessary in some cases to supplement the perfusion medium withsodium L-lactate, for example, between about 1 to 7 grams/L so that thecontrol strategy did not break down.

Additional considerations regarding choosing the levels of nutrients andglucose in the perfusion medium were necessary. For example, during theearly phases of the culture it was advantageous to quickly expand thecell mass and thus, maintain high growth rates. To keep growth rateshigh, comparatively high rates of perfusion were required to removegrowth inhibitors other than L-lactate.

According to this subject technology, Hi-end pH Control of Perfusion orHIPCOP allowed the cells to determine their rate of perfusion whichultimately depended upon their rate of glucose consumption. At any onepoint in time, the volume of perfusion medium being delivered to theculture was dependent upon the glucose concentration in the perfusionmedium. A higher concentration of glucose in the perfusion mediumcorresponds to a lower volume of perfusion medium being delivered. Forthis reason, there is value in having a lower concentration of glucosein the perfusion medium when high perfusion rates are preferred, and ahigh concentration of glucose in the perfusion medium when lower ratesof perfusion are preferred. Since many operational parameters (oxygentransfer, carbon dioxide removal, etc.) limit the final peak viable celldensities that can practically be maintained in a large-scale perfusionbioreactor, there may be value in restricting cell growth after theinitial expansion phase. When restricting additional cell division isdesirable, it may be useful to utilize a perfusion medium with a higherconcentration of glucose which would slow the perfusion rate and allowhigher levels of inhibitory molecules to accumulate in culture.

According to this subject technology, glucose concentration used in thebasal medium into which the cells are first inoculated is important. Ifthe initial glucose level is too high, it is possible the cells mightgenerate too much lactate early on in culture which would slow growthbefore perfusion could begin. With CHO cell culture, for example,initial glucose concentrations between about 2 to 4 grams/L range may beused with this subject technology.

Another consideration regarding the present subject technology is theimportance of the ratio of glucose to other nutrients (principally aminoacids) in the perfusion medium. To sustain cell growth and/orrecombinant protein production rates, this ratio must be balanced suchthat the quantity of amino acids delivered to the culture is neither toohigh nor too low. It is possible that a different ratio of glucose toamino acids, likely lower, is necessary when cell biomass production isslower. Alternatively, a concentrated slow feed of pure amino acids orpure glucose could compensate for inaccurate approximations of theproper ratio of glucose to amino acids in the perfusion medium. If apure glucose addition is necessary, this addition may need to be tied tothe pH controller/sensor in an identical manner as the perfusion mediumpump.

Thus, in an aspect, the subject technology relates to a perfusionculture process, including: (a) monitoring pH in a cell culture in aperfusion bioreactor with a pH sensor; (b) delivering fresh medium andremoving permeate when the pH is above a predetermined value; and (c)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value. In one or more embodiments related,directly or indirectly, to this aspect to each other, the fresh mediumincludes L-lactate; the L-lactate is present in the fresh medium in anamount of about 0.1 g/L to 7.0 g/L; the L-lactate is present in thefresh medium in an amount of about 1 to 4 g/L; the L-lactate is presentin the fresh medium in an amount of about 1 to 3 g/L; the L-lactate ispresent in the fresh medium in an amount of about 1 to 2.5 g/L;alternatively or in addition to L-lactate, additional sodium bicarbonateis added to the perfusion bioreactor in an amount of about 1 to about2.5 g/L such that the total sodium bicarbonate in the culture medium isabout 2 to about 5.5 g/L and wherein the additional sodium bicarbonateis delivered to the perfusion bioreactor when the pH is above thepredetermined value; the additional sodium bicarbonate is added to theperfusion bioreactor such that 1 molar carbonate enters the perfusionbioreactor at a rate of 8.7 mL per 1 liter of perfusion medium utilized;alternatively or in addition to L-lactate, any other physiologicallyacceptable base such as sodium carbonate, potassium carbonate, or thelike is added to the perfusion bioreactor in an amount that wouldprovide an upward influence on the pH in a manner similar to that whichwould occur by the addition of L-lactate; the fresh medium includes: (a)glucose; (b) L-lactate and/or additional sodium bicarbonate; and (c)amino acids; the fresh medium includes: (a) between about 0.5 to about40 g/L glucose; (b) between about 0.1 to about 7 g/L L-lactate and/orbetween about 2 to about 5.5 g/L sodium bicarbonate; and (c) amino acidsin a mole-of-glucose-to-mole-of-amino-acids ratio of between about 0.25to about 1.0; the fresh medium includes glucose in an amount equal toabout 70 mM of amino acids and about 5.3 grams of glucose per liter ofmedium; the fresh medium includes glucose at an amino-acids-(inmM)-to-glucose-(in g/L) ratio selected from the group consisting ofabout 60 to about 4.2; about 90 to about 8; about 100 to about 12; about120 to about 13; about 240 to about 42 and about 380 to about 70 perliter of medium; the predetermined pH value is about pH 7 or is about6.8 to about 7.4; a measurement of glucose concentration in the cellculture or addition of glucose to the cell culture by a glucose pump isnot required.

In an embodiment, during the growth phase, a perfusion medium with aratio of 70 mM of amino acids to 5.3 grams of glucose per liter or60:4.2, respectively, is used. In conditions of slower growth, ratios of90:8, 100:12, 120:13, 240:42 and 380:70 (mM amino acids:grams/L glucose)is used.

The subject technology's ‘cell-controlled’ perfusion system hassignificant advantages including decreasing the volumes of perfusionmedium required, reducing the burden on cell retention systems, e.g.,less filter area, small device size, more efficient separations at lowerflow rates, etc., minimizing or eliminating the accumulation of thesodium and lactate ions in the culture, and minimizing osmotic strengthincreases. The improved culture conditions additionally allow for fastercell growth rates that are closer to maximum growth rates. The lowosmotic strength and low levels of lactate is particularly valuable ifthe culture is to be transitioned in the final steps to a fed-batchculture where there is no simple method for the reduction of accumulatedions. The cell-controlled aspect of the subject technology means thatthe cells will ‘self-start’ the perfusion as well as self-control therate of perfusion ramp up. Such ‘cell-controlled’ perfusion may allowfor near instantaneous minor corrections in perfusion rate that isdesirable due to minor perturbations in the environment of the culture.In many conventional perfusion bioreactors the perfusion rate isincreased based upon a cell density being reached or an inhibitorymetabolite reaching a pre-determined concentration. The need forsampling of the bioreactor might be reduced in a HIPCOP controlledperfusion system as the perfusion rate is controlled continuously by therise in pH near instantaneously signaled by the cell metabolism. Thisopportunity to self-correct is particularly useful if the HIPCOP controlis to be used for a long-duration continuous perfusion culture.Additionally, such a ‘cell-controlled perfusion rate’ process requiresless time for process development as the optimal perfusion rates fordelivery of glucose will be determined by the cells themselves.

Alternatively or in addition, the HIPCOP or ‘cell-controlled perfusionrate’ process of the subject technology relies on little or no glucosemeasurements, which lowers the dependency of the culture process on suchvalues and thus simplifies the process to a large extent. Moreover, theHIPCOP process of the subject technology does not include a pump fordelivering highly concentrated glucose solution to the bioreactor. Inaddition, the HIPCOP process uses smaller volumes of perfusion medium(generally less than 2 reactor volumes total during the cell expansionphase) than a typical conventional perfusion processes, which makes theprocess more advantageous over the conventional processes.

Thus, in an embodiment, the subject technology relates to HIPCOP orcell-controlled perfusion culture process which includes steps of (a)monitoring pH in a cell culture with a pH sensor; (b) delivering freshmedium and removing permeate when the pH is above a predetermined value;(c) and deactivating the medium delivery and the permeate removal whenthe pH is below the predetermined value. This process is different from,for example, monitoring pH in a cell culture with a pH sensor;delivering a fresh medium and removing permeate when the pH is below apredetermined value; and deactivating the medium delivery and thepermeate removal when the pH is above the predetermined value. Thelatter process is based on the notion that if large amounts of lacticacid are being produced and the pH falls, the perfusion pumps are turnedon. If the incoming medium is of high pH, the addition of medium willindeed push the pH of the culture higher, but it will also add an excessof glucose, trigger more lactate production, continuing in a ‘viciouscycle’ that will end up using large amounts of perfusion medium to flushout lactate. The HIPCOP process of the subject technology does not havethese limitations.

The present subject technology could also be utilized for an N-1 (orseed) bioreactor in which perfusion is used to increase the cell densityto provide a high-cell density inoculum of optimum health to theproduction bioreactor. The present subject technology could also be usedfor a production reactor either as part of a continuous perfusionculture, or during a short time span of perfusion (in the productionreactor) prior to conversion to a conventional fed-batch mode ofoperation.

It is advantageous to expand cells quickly to very high density using ashort duration of perfusion (continuous culture with cell retention),and then complete the production culture as a simple fed-batchbioreactor. The perfusion operation of such a culture would likelycontinuously ‘ramp up’ the perfusion rate as the cell density increasedto keep the cells growing at a near exponential rate. While continuingto add complexity, there is also value in performing a short duration ofperfusion with a dilute medium (which will add nutrients and removewaste products simultaneously) followed by perfusion at much lower rateswith a highly concentrated medium (no longer efficiently flushing outinhibitors, but still adding sufficient nutrients to support productionof the product of interest). Such a culture would allow for very fastgrowth to high density, and then allow for high productivity, andgenerally minimize the length of the culture such that it is of similarlength to a more conventional fed-batch culture.

Thus, for a hybrid perfusion/fed-batch culture process, the process maybe carried out in the following manner (numbers used for this exampleare taken from the process data presented from cell line A as in FIG.6-8 ). Upon production bioreactor inoculation, a batch comprising a highinoculation density of approximately 5×10⁶ cells/mL is cultured in aninitial medium of 4 g/L glucose. After one day and for the following 3days, the batch is switched to a continuous perfusion process wherecells “self-control” the perfusion ramp up. Approximately 2.5 g/L sodiumL-lactate is present in the perfusion medium. Perfusion occurs withapproximately 60 L of working volume of which approximately 160 L totalof perfusion medium is required. After three days, the process isswitched to a standard fed-batch process and feeds continue until thebioreactor reaches 100 L on the day 12 of the culture. By using thishybrid perfusion/fed-batch culture process, volumetric productivity ofequal to or greater than 1 gram cells/L/day has been achieved.Additionally, if a typical conventional perfusion ramp up was used, itis likely that 2-3 times more perfusion medium would have been requiredand additional filter area for the cell retention system may have alsobeen required to achieve similar results.

In an embodiment relating to the hybrid perfusion/fed-batch cultureprocess, the perfusion process is implemented first for the cell cultureand the fed batch process is implemented second. In another relatedembodiment, the perfusion process starts first and lasts for 1 to 12days and the fed-batch process follows the perfusion process and lastsfor additional 1 to 12 days. In an exemplary embodiment, the perfusionprocess lasts for 3 to 5 days (typically starting automatically within24 hours of inoculation) and the fed-batch process follows the perfusionprocess and lasts for additional 9 to 11 days. The advantages of thishybrid system are very high volumetric productivity, fits in standardfed-batch window, and fits in existing facilities (e.g., singleharvest). The HIPCOP in this system allows cells to control their ownperfusion rates; it adjusts to minor process deviations and lactate,ammonia, osmolality remain very low just prior to start of fed-batch.

Another embodiment of the subject technology relates to a continuousperfusion bioreactor which consists of two significantly differentphases. The first phase includes the initial continuous phase asmentioned above (ramp up of cell density and perfusion rate usingHIPCOP) utilizing a comparably dilute medium, followed by a second phaseof perfusion in which the perfusion rates are significantly reduced byutilization of a highly concentrated perfusion medium.

Another embodiment of the subject technology relates to the use of adiluent liquid that is added to the bioreactor during the later stagesof the perfusion culture when the concentrated perfusion medium is beingadded to the bioreactor. In one embodiment, such a diluent liquid is asolution of saline of appropriate concentration (e.g., 2.0 g/L sodiumbicarbonate, 2.4 g/L polyvinyl alcohol, 20 mM potassium chloride, and 80mM sodium chloride). Because the long-distance transport of liquidnutrient medium can incur many difficulties (cost of transport,maintenance of sterility, temperature control) there is significantvalue in using highly concentrated perfusion medium in an industrialsetting. Such medium allows for perfusion rates as low as 0.05-0.30reactor volumes per day for the concentrated perfusion medium. In arelated embodiment, in such a bioreactor system it necessary to flushproduct material out of the bioreactor, particularly if a continuousdownstream process is linked directly to capture the continuouslydelivered upstream harvest material, and if the protein being producedis highly labile. Additionally, in order to avoid an excessively largedownstream it may also be advantageous to control the mass per day ofproduct entering the downstream process within a small range. This canalso be facilitated by manipulating the flow rate of the diluent, e.g.,saline. It is also advantageous to maintain the osmotic strength of thebioreactor environment close to the physiological range of 250-350 mOsm.Cells often produce extraneous metabolites that increase the osmoticstrength of the culture and can negatively impact culture health andcellular productivity. Additionally, since the perfusion medium in thelate stage of the continuous culture is extremely concentrated and theremay be variability of cellular uptake rates of amino acids, some aminoacids might accumulate in the bioreactor, potentially also negativelyimpacting the culture environment due to toxicity or merely due to theircontribution to the increase in osmotic strength. Both of theseobjectives (flushing product material or accumulating unconsumednutrients out of the bioreactor, and maintaining appropriate cultureosmotic strength) might be facilitated by a feed to the bioreactor of asolution of saline at the optimal concentration (e.g., between about 250to 350 mOsm/kg or between about 0 to 250 mOsm/kg). Such a diluent couldbe continuously added to the bioreactor and its saline content could becontinuously adjusted by addition of sterile water (again with the goalof potentially minimizing the need to transport/ship large volumes ofliquid) such that the near optimal culture environment could bemaintained at any time point of the culture. Feed-back control usingin-line or off-line analysis of osmotic strength and productconcentration could also facilitate the addition of the proper amountand concentration of the salt and water solutions. Furthermore, thesaline or diluent solution could be near saturation with sodiumchloride, or more preferentially have a mixture of sodium chloride andpotassium chloride near saturation so that additional potassium can besupplied as a nutrient to the culture, or that a more physiologicallyappropriate ratio of potassium to sodium ion might be maintained in theculture.

EXAMPLES Example 1 The Application of HIPCOP Technology at the 1-2 LiterScale

Multiple tests using HIPCOP (high-end pH control of perfusion) have beenperformed. This process was advantageous during the ramp up of perfusionas cell density increased. L-lactate was kept low and osmotic strengthwas maintained in an optimal range. FIGS. 3A-E compare two continuousperfusion cultures, one using HIPCOP and one maintained undernon-limiting glucose conditions, i.e., additional glucose was added tothe culture to maintain between about 0.5-3.5 g/L glucose. Both cultureshad identical set points and dead bands for pH, dissolved oxygen, andtemperature control. Perfusion started automatically between days 2 and3 when the glucose in the HIPCOP bioreactor fell to a low level, lacticacid was taken up by the cells, and the bulk pH rose to trigger thestartup of the perfusion medium addition and permeate removal pumps. Therate of addition of perfusion medium to the two cultures was identical,and was ramped up at the same rate controlled by the otherwise identicalHIPCOP culture. The rate of cell growth was nearly identical in the twocultures despite the fact that the HIPCOP bioreactor maintained a nearlyzero glucose concentration while the perfusion was occurring. Cellviabilities were also similar but slightly lower for the HIPCOPcondition (FIG. 3B).

Because the non-limited glucose condition produced an excess of lacticacid and basic titrant was automatically added to maintain pH, aslightly higher overall volume of fluid (9% by volume more fluid) waspassing through the cell mass when compared with the HIPCOP condition.While the cell densities and growth rates were very similar, far moreL-lactate was produced in the non-limited glucose culture, and a farhigher osmotic strength was reached as the culture progressed (see FIGS.3D and 3E).

FIGS. 4A-C demonstrates the application of the HIPCOP technology toanother CHO cell line (cell line B). Again the cells divided at a growthrate near the maximum growth rate for the CHO cell line. Again theL-lactate and osmotic strength of the culture was suppressed and theculture environment was near optimum for continued operation.

A specific example of fresh perfusion medium that may be used accordingto this subject technology comprises 8 g/L glucose, 2.5 g/L sodiumL-lactate (i.e., 22 mM L-lactate ion) and 90 mM amino acids.

Example 2 The Application of HIPCOP Technology at the 70 Liter Scale

The following data shows that the technique of HIPCOP can easily beimplemented at the 70-liter scale with similar results to that of the1-2 liter scale.

The techniques used to develop the subject technology at the 1-2 literscale were implemented in a 150-L stainless steel bioreactor systemfitted with a scaled up hollow fiber filtration module and recirculationloop. The hollow fiber filtration module had a surface area of 2.55meters square and a 0.2 micron pore size. Liquid was recirculatedthrough the external perfusion loop at 8-9 liters per minute and theworking volume of the bioreactor was 70 liters. At the pilot scale theperfusion medium composition was 90 millimolar amino acids, 8 g/Lglucose, and 2.5 g/L sodium L-lactate (i.e., 22 mM L-lactate ion) with afinal osmolality of 345 mOsm.

FIGS. 5A-C show the application of the HIPCOP technology with cell lineA at the 70-Liter scale. Growth rates and cell viability were slightlylower at the large scale compared to the previous data from 1-2 Lbioreactors. Additional optimization (to minimize cell shear damage) ofthe equipment used in the perfusion loop of the large scale bioreactormay be possible. The inoculation density in the pilot scale experimentwas significantly higher than the previous data for cell line A from the1-2 L bioreactors. The final cell densities reached at the pilot scalewere somewhat higher than those obtained at the small scale. L-lactateand the resulting osmolality of the culture at the pilot scale were wellcontrolled as in the small scale experiments. As expected as a result ofthe method of the invention, glucose levels quickly dropped and remainedvery low throughout the HIPCOP perfusion. It was not necessary tomanually adjust the perfusion rate during the experiment at the pilotscale. Only one maximum pump speed was set and the pumps (both the feedand permeate pumps) were turned on and off by the high-end pHcontroller/sensor system to effectively ramp up the perfusion rate ofthe culture.

Example 3 The Application of HIPCOP Technology to Additional Cell Linesin a Hybrid Perfusion/Fed-Batch Process

Additional data for four different glutamine-synthetase CHO cell linesproducing monoclonal antibodies are shown in FIGS. 6, 7, and 8 . Alldata was generated with a nearly identical process with a continuousperfusion period of 4 days (perfusion ramp up using HIPCOP), after whichthe culture process was switched to a fed-batch process for theremainder of the 12-day culture. As such, the cells were cultured undera hybrid (i.e., HIPCOP perfusion/fed-batch) process. The perfusionmedium composition was identical to that used in example 2, 90millimolar amino acids, 8 g/L glucose, and 2.5 g/L sodium L-lactate(i.e., 22 mM L-lactate ion) with a final osmolality of 345 mOsm. Asshown in FIG. 6 , in this hybrid process, the cell lines reached amaximum cell density of between 67-85×10⁶ cells/mL on days 5 and 6 witha high percentage viability of about 87 to about 97% for the entirelength of the process. In some pilot-scale (˜100 L) hybrid processes,cultured cells reached a density of over 95×10⁶ cells/mL (data notshown). Titers between 6 and nearly 12 grams/liter were reached with thefour different cell lines are summarized in FIG. 6E. FIG. 7 compares thetiters achieved with the four CHO cell lines in an optimized fed-batchprocess with the titers achieved when applying the hybrid perfusionfed-batch process utilizing HIPCOP. FIG. 8 shows the very modest volumesof medium used during the HIPCOP perfusion stage of the hybrid perfusionfed-batch process. The reactor volumes are calculated based on the finalvolume of bioreactor that would be necessary to accommodate the feedsthat occur during the fed-batch portion of the bioreactor operation(volume of feeds are also shown in FIG. 8 ). These results show thatapplying the HIPCOP technology to optimized late-stage fed-batchprocesses can more than double the productivity with minimal developmentefforts.

Example 4 The Application of HIPCOP Technology to a Continuous PerfusionProcess

FIG. 9A tracks the viable cell density of a 2 L bioreactor with CHO cellline A in a continuous perfusion process that uses the HIPCOP technologyto control perfusion for the first six days, and then reverts to acontinuous perfusion with manually-controlled, but comparatively low,perfusion rates beyond day 6. The perfusion medium used during the firstsix days was identical in make up to that used in example 3 (90millimolar amino acids, 8 g/L glucose, and 2.5 g/L sodium L-lactate(i.e., 22 mM L-lactate ion) with a final osmolality of 345 mOsm). Fromday 6 to day 17 the culture was perfused using a highly concentratedperfusion nutrient solution of 600 millimolar amino acids (90grams/liter glucose, 0 g/L sodium L-lactate, final osmolality ofapproximately 1300 mOsm/kg) and a saline diluent (2.0 g/L sodiumbicarbonate, 2.4 g/L polyvinyl alcohol [a shear protectant], 20 mMpotassium chloride, and 80 mM sodium chloride, pH of 7.10, finalosmolality of 250 mOsm/kg) in varying ratios as indicated in FIG. 9F. Inlater experiments (data not shown) it was determined that similarresults could be obtained when the sodium bicarbonate and the polyvinylalcohol were removed from the saline diluent. In this case the levels ofsodium chloride were increased to achieve a similar final osmoticstrength of 250 mOsm/kg (20 mM potassium chloride and 105 mM sodiumchloride) for the diluent. To minimize volumes of liquid that might needto be transported, at the large scale it might be optimal to use anearly saturated solution of potassium chloride and sodium chloride witha molar ratio of approximately 1:5 respectively and dilute as necessarywith additional water.

Viable cell densities in this system reached extremely high levels, andwere sustained over 100×10⁶ cells/milliliter for several days (FIG. 9A).Cell viabilities were also high as shown in FIG. 9B. L-lactate levelswere well controlled and remained at or below 2 grams/liter during theexpansion phase of the culture as the HIPCOP perfusion was operating asshown in FIG. 9C. Osmolality was also well controlled as shown in FIG.9D. The control of osmolality was facilitated by the variable rateaddition of the saline diluent solution that was initiated on day 6 andramped up relative to the perfusion rate of the concentrated nutrientperfusion medium towards the conclusion of the experiment (FIG. 9F). Therates of perfusion achieved by adding the saline diluent andconcentrated perfusion nutrient solution were manipulated in an effortto simultaneously add sufficient nutrients to sustain the cell biomassand produce the protein of interest, to facilitate the removal of theprotein of interest from the bioreactor through the cell retentionhollow-fiber filtration device and into the continuous downstreamprocess at a controlled rate within a reasonably narrow band of totalmass/day, and also to maintain the osmotic strength of the culture nearto an optimal window of 250-350 mOsm.

FIG. 9E shows the titer achieved in the bioreactor and that in thepermeate leaving the bioreactor system through the hollow-fiberfiltration device. Likely due to a build-up of protein on the surface ofthe micro-filtration hollow-fiber module, the concentration of antibodyin the bioreactor becomes higher than that leaving in the permeate. Thisselective concentration of the protein of interest inside the bioreactormay be undesirable if a continuous downstream process is used. There maybe minor modifications to the cell retention system which could be usedto eliminate or minimize this problem (larger microfiltration pore size,larger filter area, alternative microfiltration materials ofconstruction, more aggressive tangential flow, back-flushing of thefilter) or methods of cell retention that are less prone or imperviousto production retention might be employed (spin filters, acoustic wavecell settling devices, inclined-plane gravity cell-settling devices).

FIG. 10 list several important parameters for the continuous perfusionprocess (high-intensity, low-volume perfusion). The table lists theoverall volumetric productivity (grams/Liter of reactor volume/day) ofthe process, and the total volumes of the various perfusion mediautilized during the entire experiment (in reactor volumes). As evidencedby the data in the table, the volumetric productivity is very high andthe overall volumes of perfusion medium are very modest for thecontinuous perfusion process which uses HIPCOP during expansion (days0-6) and later uses the saline diluent to control osmotic strength whenthe highly concentrated perfusion medium is utilized from days 6-17.FIG. 11 compares the volumetric productivity of both the hybridperfusion fed-batch (second bar from the left) and the continuousperfusion process (third bar from the left), both processes using HIPCOPduring the cell expansion phase, to the optimized fed-batch process(first bar on the left) that had been developed for model cell line A.As seen in the figure, the continuous perfusion process (high intensitylow volume perfusion) using HIPCOP during the expansion phase has nearlya 4-fold increase in volumetric productivity when compared to theoptimized fed-batch process.

In the high intensity low volume perfusion process, a diluent liquid isadded to the bioreactor during the later stages of the perfusion culturewhen the concentrated perfusion medium is being added to the bioreactor.For example, the diluent liquid (a solution of saline) of appropriateconcentration (e.g., 2.0 g/L sodium bicarbonate, 2.4 g/L polyvinylalcohol, 20 mM potassium chloride, and 80 mM sodium chloride) was addedto the bioreactor. Using a diluent liquid (e.g., saline) in combinationwith the concentrated perfusion medium allows for perfusion rates as lowas 0.05-0.30 reactor volumes per day for the concentrated perfusionmedium. The addition of the diluent will also facilitate the flushing ofthe product material out of the bioreactor, particularly if a continuousdownstream process is linked directly to capture the continuouslydelivered upstream harvest material, and if the protein being producedis highly labile. Additionally, in order to avoid an excessively largedownstream it is also advantageous to control the mass per day ofproduct entering the downstream process within a small range. This canalso be facilitated by manipulating the flow rate of the diluent, e.g.,saline. The addition of the diluent can also advantageously facilitatethe control over or the maintenance of the osmotic strength of thebioreactor environment close to the physiological range of 250-350 mOsm.

Example 5 Use of HIPCOP Technique with High Sodium Bicarbonate inPerfusion Medium and a DG44 Derived CHO Cell Line Expressing aRecombinant Protein of ˜130 kDa

The method of using additional sodium bicarbonate in the perfusionmedium in place of sodium-L or sodium D/L lactate was tested with anadditional cell line, cell line “E”. The perfusion medium contained 90millimolar amino acids, 8 grams/L glucose, 10 millimolar glutamine, 3.87grams/L sodium bicarbonate (which is approximately 1.87 grams/Ladditional that would have been in the perfusion medium if sodiumL-lactate were being used at 2.5 grams/L), with a final osmotic strengthof 330 mOsm/kg. The initial basal medium in this experiment consisted of120 millimolar amino acids, 6 grams/L glucose, 10 millimolar glutamine,and 10 mg/L recombinant insulin. Cells were inoculated at approximately10×10⁶ viable cells/ml. Cell growth was very fast and the perfusion wasinitiated by the cells at approximately 23 hours after inoculation.Perfusion continued for approximately 29 hours over which time theculture used a total of 0.85 reactor volumes of perfusion medium. Theperfusion rate in the last 4 hours of perfusion was approximately 1.4reactor volumes per day.

As in the case with the previous examples, the HIPCOP method ofcell-controlled perfusion worked well with this cell line. Cells grew to47×10⁶ viable cells/ml within 52 hours after inoculation (FIG. 12A).FIG. 12B shows that glucose was quickly consumed and then maintained ata low concentration using the subject technology. Additionally, FIG. 12Bshows that lactate was controlled at a low level, ending at about 1.3grams/liter. FIG. 12C shows that the osmotic strength of the culture wasalso maintained very close to a physiologically ideal range near 300mOsm/kg throughout the time the perfusion was being performed.

Example 6 Use of HIPCOP Technique with High Sodium Bicarbonate inPerfusion Medium and a Glutamine-Synthetase CHO Cell Line Expressing aRecombinant Immunoglobulin G

The method of using additional sodium bicarbonate in the perfusionmedium in place of sodium-L or sodium D/L lactate was tested with anadditional cell line, cell line “B”. The perfusion medium contained 90millimolar amino acids, 10 grams/L glucose, 3.87 grams/L sodiumbicarbonate (which is approximately 1.87 grams/L additional than wouldhave been in the perfusion medium if sodium L-lactate were being used at2.5 grams/L), with a final osmotic strength of 366 mOsm/kg. The initialbasal medium in this experiment consisted of 120 millimolar amino acids,4 grams/L glucose. Cells were inoculated at approximately 1.2×10⁶ viablecells/ml. Perfusion was initiated by the cells at approximately 2.3 daysafter inoculation. Perfusion continued for approximately 3.7 days overwhich time the culture used a total of 2.09 reactor volumes of perfusionmedium. The perfusion rate in the last 4 hours of perfusion wasapproximately 1.23 reactor volumes per day.

As in the case with the previous examples, the HIPCOP method ofcell-controlled perfusion worked well with this cell line. Cells grew to57×10⁶ viable cells/ml within 5.8 days after inoculation (FIG. 13A).FIG. 13B shows that glucose was quickly consumed and then maintained ata low concentration using the subject technology. Additionally, FIG. 13Bshows that lactate was controlled at a low level, ending at about 1.47grams/liter. FIG. 13C shows that the osmotic strength of the culture wasalso maintained very close to a physiologically ideal range near 300mOsm/kg throughout the time the perfusion was being performed.

Example 7 Use of HIPCOP Technique with Sodium Carbonate Feed and aGlutamine-Synthetase CHO Cell Line Expressing a RecombinantImmunoglobulin G

This is an example of the use of HIPCOP to control the perfusion rate ofa ‘sustainable’ continuous perfusion bioreactor. The example starts witha perfusion reactor operating at a near steady-state condition withHIPCOP allowing the cells to control their own perfusion rate withsodium-L-lactate in the perfusion medium. A change is then made to thecomposition of that perfusion medium, removing sodium-L-lactate.

The method of using a separate carbonate feed in place of sodium-L orsodium D/L lactate in the perfusion medium was tested with cell line“B”. The data for two steady-states are shown. The perfusion medium forsteady-state 1 contained 110 millimolar amino acids, 10 grams/L glucose,2.6 g/L sodium lactate, 2.0 grams/L sodium bicarbonate, with a finalosmotic strength of 405 mOsm/kg. The perfusion medium for steady-state 2contained 110 millimolar amino acids, 12.1 grams/L glucose, 2.0 grams/Lsodium bicarbonate, with a final osmotic strength of 403 mOsm/kg. Notethat the total carbon source with respect to glucose and lactate werekept approximately the same between the perfusion media used during bothsteady-states. Perfusion was maintained by the cells and a cell-bleedwas adjusted once daily to maintain the viable cell-density at a targetof 40×10⁶ viable cells/ml. The continuous steady-state perfusion ratethat the cells have ‘determined’ is approximately 1.0 reactor volumesper day. During steady-state 1, no carbonate was added. Duringsteady-state 1 the cells are clearly consuming a significant fraction ofthe lactate that is entering in the perfusion medium since the residuallevels of lactate in the bioreactor are lower than that in the perfusionmedium. They presumably are consuming the lactate as lactic acid. Theconsumption of lactic acid by the cells maintains a continuous upwardinfluence on the pH of the culture and allows the HIPCOP technology tofunction properly. When steady-state 2 is initiated this continuousupward influence on the pH of the culture is now instead providedthrough the semi-continuous addition of a 1 molar carbonate solution (inthis case the carbonate is a mixture of sodium and potassium carbonatein the molar ratio of 0.94 sodium:0.06 potassium) at a rate ofapproximately 8.7 mL per liter of bioreactor volume. Therefore theaddition of 1 molar carbonate enters at a ratio of 8.7 mL per 1 liter ofperfusion medium utilized.

As in the case with the previous examples, the HIPCOP method ofcell-controlled perfusion worked well with this cell line during bothsteady-states. The average perfusion rate and cell-specific perfusionrate during each steady-state were 1.0 reactor volumes per day and 25picoliters/cell/day, respectively (FIGS. 14C & 14D). FIG. 14B shows thatduring both steady-states, lactate was maintained within a range of1.5-1.8 g/L. FIG. 14E shows that the osmotic strength of the culture wasmaintained in a physiologically ideal range near 300 mOsm/kg. There wasa slight change in the steady-state osmotic strength of the culture fromsteady-state 1 to steady-state 2, this may have been due to theadditional sodium entering the bioreactor in the 1 molar carbonatesolution that was not completely accounted for when the perfusion mediumcomposition was designed and prepared (a small fraction of the sodiumchloride normally used in the perfusion medium preparation should havebeen excluded).

While in the current example a mixture of sodium and potassium carbonateadded continuously were used to provide the upward pressure on pH thatthe consumption of lactic acid from the perfusion medium would havesupplied if sodium-L-lactate were in the perfusion medium, presumablyany appropriate non-toxic basic substance added in a continuous orsemi-continuous manner to the culture could provide the same effect.Examples of such bases could include sodium or potassium hydroxide,among many others.

While the subject technology has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofordinary skill in the art that various changes and modifications can bemade to the claimed invention without departing from the spirit andscope thereof. Thus, for example, those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are considered to bewithin the scope of the subject technology, and are covered by thefollowing claims.

INDUSTRIAL APPLICABILITY

The device and methods disclosed herein are useful for perfusionbiomanufacturing, and thus for improving industrial methods formanufacturing recombinant, therapeutic proteins.

What is claimed is:
 1. A method for achieving rapid cell growth in acontinuous perfusion culture process, comprising: (a) monitoring pH in acell culture with a pH sensor; (b) delivering fresh medium and removingpermeate when the pH is above a predetermined value; and (c)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value.
 2. The method of claim 1, wherein thefresh medium comprises: (a) between about 0.5 and 40 g/L glucose; (b)between about 0.1 to 7.0 g/L L-lactate; and (c) amino acids in a ratioof between about 0.25 and 1.0 of mole glucose to mole amino acids. 3.The method of claim 1, wherein the rapid cell growth is achieved within4 days.
 4. The method of claim 1, wherein the measurement of glucoseconcentration in the cell culture or additional of glucose to the cellculture by a glucose pump is not required.
 5. The method of claim 2,wherein the L-lactate is present in the fresh medium in an amount ofabout 1 to 4 g/L.
 6. The method of claim 3, wherein the L-lactate ispresent in the fresh medium in an amount of about 1 to 2.5 g/L.
 7. Themethod of claim 3, wherein the L-lactate is sodium L-lactate orpotassium L-lactate.
 8. The method of claim 2, wherein the amino acidsand glucose are provided in an amount equal to 70 mM amino acids and 5.3grams of glucose per liter of medium or at ratios selected from thegroup consisting of 60:4.2; 90:8; 100:12; 120:13; 240:42 and 380:70 mMamino acids:grams/L glucose per liter of medium.
 9. The method of claim1, wherein the predetermined value is about pH
 7. 10. A hybrid cultureprocess, comprising: a continuous perfusion culture process of as thefirst culture process, wherein the continuous perfusion culture processcomprises (a) monitoring pH in a cell culture with a pH sensor; (b)delivering fresh medium and removing permeate when the pH is above apredetermined value; and (c) deactivating the medium delivery and thepermeate removal when the pH is below the predetermined value.
 11. Thehybrid culture process of claim 10, wherein the continuous perfusionculture process is followed by a fed-batch process.
 12. The hybridculture process of claim 10, wherein the continuous perfusion cultureprocess is followed by a second continuous perfusion culture process.13. The hybrid culture process of claim 12, wherein the secondcontinuous perfusion culture process comprises the steps of: (a) addinga concentrated medium; and (b) adding a diluent.
 14. The hybrid cultureprocess of claim 13, wherein the concentrated medium comprises 600millimolar amino acids, 90 grams/liter glucose, and 0 g/L sodiumL-lactate.
 15. The hybrid culture process of claim 13, wherein thediluent is selected from the group consisting of saline and water. 16.The hybrid culture process of claim 15, wherein the saline or waternormalizes osmotic strength of culture medium to between about 0 to 250and/or 250 to 350 mOsm/kg.
 17. The hybrid culture process of claim 15,wherein the saline comprises 2.0 g/L sodium bicarbonate, 2.4 g/Lpolyvinyl alcohol, 20 mM potassium chloride, and 80 mM sodium chloride.18. The hybrid culture process of claim 10, wherein the hybrid cultureprocess results in volumetric productivity of greater than 1 gramcells/L/day.
 19. The hybrid culture process of claim 10, wherein thehybrid culture process is performed in a culture volume of about 50 andabout 150 L.
 20. The hybrid culture process of claim 19, wherein theculture volume is about 70 L.
 21. A bioreactor for conducting acontinuous perfusion culture process, wherein the continuous perfusionculture process comprises the steps of: (a) monitoring pH in a cellculture with a pH sensor; (b) delivering fresh medium and removingpermeate when the pH is above a predetermined value; and (c)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value.
 22. A perfusion culture process,comprising: (d) monitoring pH in a cell culture in a perfusionbioreactor with a pH sensor; (e) delivering fresh medium to theperfusion bioreactor and removing permeate when the pH is above apredetermined value; and (f) deactivating the medium delivery and thepermeate removal when the pH is below the predetermined value.
 23. Amethod for achieving rapid cell growth in a perfusion culture process,comprising: (d) monitoring pH in a cell culture in a perfusionbioreactor with a pH sensor; (e) delivering fresh medium to theperfusion bioreactor and removing permeate when the pH is above apredetermined value; and (f) deactivating the medium delivery and thepermeate removal when the pH is below the predetermined value.
 24. Abioreactor for conducting a continuous perfusion culture process,wherein the continuous perfusion culture process comprises the steps of:(d) monitoring pH in a cell culture in a perfusion bioreactor with a pHsensor; (e) delivering fresh medium to the perfusion bioreactor andremoving permeate when the pH is above a predetermined value; and (f)deactivating the medium delivery and the permeate removal when the pH isbelow the predetermined value.
 25. A protein of interest produced by amethod comprising: (a) culturing cells comprising a gene that encodesthe protein of interest in a perfusion cell culture bioreactor underconditions that allow production of the protein of interest comprising:i. monitoring pH in the cell culture bioreactor with a pH sensor; ii.delivering fresh medium and removing permeate when the pH is above apredetermined value; and iii. deactivating the medium delivery and thepermeate removal when the pH is below the predetermined value. (b)harvesting the protein of interest from the cell culture bioreactor.